1) Field of the Invention
The present invention relates generally to diagnostic methods in histology and cytology, and more specifically to diagnostic methods for the detection of cells undergoing apoptosis.
2) Related Technology
Apoptosis is an intrinsic program of cell self-destruction inherent in eukaryotic cells. Apoptosis also plays a role in cell creation, and it is becoming increasingly apparent that the processes of cellular creation and destruction are linked by common signaling mechanisms. For example, up-regulation of one process may be accompanied by down-regulation or up-regulation of the other. Thus apoptosis may play an important role in understanding various diseases.
For example, a variety of diseases and other pathological conditions are linked to dysregulated apoptosis in a particular tissue or organ. These include, for example, general conditions related to tissue rejection, immune/inflammatory responses, ischemia and injury, cardiovascular diseases such as dilated or ischemic cardiomyopathy, myocarditis and atherosclerosis, neurodegenerative disorders such as ALS, Alzheimer's disease, Parkinson's disease and retinal degeneration, hepatic and pancreatic disorders related to viral infection or alcohol consumption, which can lead to development of insulin-dependent diabetes mellitus or infection with certain viruses, such as adenoviruses, influenzaviruses and human immunodeficiency virus.
Additionally, a variety of cell proliferative diseases and disorders are linked to dysregulated apoptosis. These include, for example, psoriasis, lupus and other autoimmune conditions such as Crohn's disease, Hashimoto's thyroiditis and arthritis, infection with certain viruses, such as human papillomavirus, Epstein-Barr virus and herpes simplex virus; as well as a variety of cancers, including mammary carcinomas, lymphomas, cervical, colon, and ovarian cancers, and neuroblastomas.
Because apoptosis plays such an important role in so many medical disorders, rapid and reproducible assays for apoptosis are of great interest to investigators in their attempts uncover disease pathways and to devise clinically relevant diagnostic and prognostic indicators of disease status in a patient.
Presently, the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) is one method used to detect apoptosis. The TUNEL method utilizes TdT to incorporate labeled nucleotides (dNTPs) into DNA fragments containing a 3′-hydroxyl group. The label can be detected using a variety of methodologies, e.g. immunochemical or fluorescent detection. Label detection is indicative of apoptosis. While traditional TUNEL methodologies are important, they are also very labor intensive.
Traditional TUNEL methodologies require approximately four hours of constant manual attention and manipulation of samples to process 6-12 slides. Because at least two of those slides are controls, the current methodology leaves much to be desired in terms of speed, throughput, and reproducibility. The labor costs associated with these technologies are also high.
Further, while many processes are being automated in the interest of high-throughput and reproducibility, not all labs (e.g. principle investigators at small universities, small startups, or small clinical hospitals) can house, or afford the cost of, every automated system that comes out on the market.
Thus what is needed are protocols that expand the function of existing research tools. For example, many labs have stainers for histology and immunochemical purposes. The ability to use existing staining machines to perform rapid and reproducible TUNEL assays would be greatly desired.
By way of example, research laboratories perform TUNEL assays manually at great cost in skilled labor because, as noted previously, current methodologies require essentially constant attention by highly trained workers. These same labs, however, also typically have immunochemical stainers and/or histological stainers. The ability to use existing stainers to run more tests at one time, and produce more rapid and accurate test results, would significantly decrease costs to the institution and the consumer. Similar advantages and savings would also be recognized through the use of novel systems designed specifically for automating TUNEL assays.